Rearing of aquatic species with DHA-rich prey organisms

ABSTRACT

A method of rearing aquatic species is provided, comprising feeding the aquatic species during at least part of the larval and/or post-larval state with prey organisms, such as  Artemia  or rotifers, having in their total lipid content a DHA content of at least 12 wt %. The method is suitable for the rearing of fish species such as halibut, turbot, bass, bream and flounder. The total lipid content of the prey organisms further including highly unsaturated fatty acids comprising 18:3, 18:4, 20:4, 20:5 (EPA) and 22:5 fatty acids. Also the prey organisms include 5 to 35 wt % phospholipids of the total lipid content, wherein the total lipid content is at least 20 wt % on a dry weight basis.

FIELD OF THE INVENTION

The present invention is within the field of aquaculture, in particularthere is provided a method for aquacultural rearing of aquatic specieson prey organisms enriched in highly unsaturated fatty acids (HUFAs), inparticular docosahexaenoic acid (DHA), providing high survival ratesduring the fish larval stage.

TECHNICAL BACKGROUND AND PRIOR ART

The consumption of seafood species for which there is a high consumerdemand such as salmon, trout, halibut and eel is increasing and due tothis high demand and limited natural stocks, much effort is spent ondeveloping cost effective aquacultural methods of farming such species.A particularly serious problem is to secure a high survival rate of thehatched larvae of the species being cultivated.

Expansion of the aquaculture industry requires that several problems beaddressed, one of the most significant being the difficulty of supplyinglive prey organisms which provide a nutritionally adequate feed for thelarvae. Larval fish in the wild consume a mixed population ofphytoplankton prey organisms that provide a balanced nutrition. However,collecting phytoplankton in sufficient quantities to meet the demand inaquaculture is not feasible. As an alternative, selected species of preyorganisms, in particular rotifers and Artemia species, are presentlycultivated and used as feed.

Generally however, such artificially cultivated prey organisms, althoughthey provide adequate amounts of protein and energy, have a lipidcomposition which is not adequate to cover the requirement for certainHUFAs, in particular DHA and EPA (eicosapentaenoic acid) which areessential for the optimum survival, growth and development of larvae.Specifically, it has been shown that a high content of DHA is requiredand that the ratio between DHA and EPA In the prey organisms should beat least 1:1 and preferably at least 2:1.

Currently, this problem is being addressed by cultivating the preyorganisms in the presence of enrichment compositions permitting theorganisms to be enriched in respect of these essential fatty acids.However, presently available commercial compositions for that purposesuch as products sold under the tradename Selco (TM) do not meet theabove requirements in that the DHA content is relatively low and/or theDHA: EPA ratio is not high enough. Using such compositions Artemiaenrichment levels of 3-5% DHA of total lipids have been reported (McEvoyet al. Aquaculture 163 (1998) 237-250), and 12 to 15% survival rates offish fed such Artemia (McEvoy et al. supra; Navarro et al. J. Fish Biol.43 (1993) 503-515). ). In this context, survival rates are defined assurvival percentage from first feeding through metamorphosis. Forcost-effective aquaculture production a larval survival rate of 50% andpreferably higher should be obtained.

WO 99/37166 discloses a method for the enrichment of live prey organismswith nutrients essential for fish larvae based on the use of dry soappowders of HUFAs obtained from the waste stream of marine algae oilextraction. Artemia DHA enrichment levels of about 2.7% of dry weightare disclosed, but the use in aquaculture and efficacy with respect tofish larvae survival is not disclosed.

Another material intended for use in aquaculture is described in WO99/06585. Examples disclose a DHA content of 24 wt %, but thephospholipid content is not disclosed. The material however, contains ahigh proportion of free fatty acids (about 32-37 wt %) and a highcontent of non-lipid material which may reduce the lipid uptakeefficiency of prey animals. A high content of free fatty acids isgenerally considered harmful for fish juveniles.

Neither of the two last-mentioned materials is fish-based and they lackmany HUFAs found in fish, such as EPA and other n-3 fatty acids,desirable for fish juveniles.

In a recent review by Sargent et al. (Aquaculture 179 (1999) 217-229) itis emphasized that in addition to the requirement in respect of HUFAs,fish larvae have a dietary requirement for phospholipids and it isstressed that the ideal diet for fish larvae is a diet having acomposition similar to the yolk of the eggs. According to these authorsfish egg yolk contains about 10 wt % (on a dry matter basis)phospholipids which contain about 17 wt % of DHA and about 9 wt % ofEPA. These authors conclude in their review that a problem remains withrespect to how to construct such a diet on a commercial scale fromcurrently available materials.

It has now been found that it is possible to provide enrichedaquacultural prey organisms having, in respect of HUFAs andphospholipids, a composition which is very close to that of fish eggyolk. By using the prey organisms of the invention it is possible tosecure optimum survival, growth, pigmentation and morphogenesis ofaquatic organism larvae such as halibut larvae. As demonstrated herein,the invention provides much higher survival rates during the larvalstage and increased quality parameters than previously disclosed forfish such as Halibut, thus making aquacultural rearing of manyhigh-demand fish species more economical and commercially viable.

SUMMARY OF INVENTION

The invention provides a method of rearing an aquatic species, themethod comprising feeding the aquatic species during at least part ofthe larval and/or post-larval stage with prey organisms having in theirtotal lipid content a content of DHA of at least 12 wt %.

DETAILED DESCRIPTION OF THE INVENTION

The method according to the invention, of rearing an aquatic species,comprises feeding the aquatic species during at least part of the larvaland/or post-larval stage with prey organisms having in their total lipidcontent a content of DHA of at least 12 wt %, preferably at least 15 wt%, such at least 17 wt % more preferably at least 20 wt %, such as atleast 25 wt %.

In preferred embodiments, the prey organisms also contain a significantamount of phospholipids, such as e.g. in the range of 5-35 wt % of thetotal lipid content, such as in the range of 5-25 wt % including in therange of 10-20 wt % of total lipid.

It is additionally preferred that the prey organisms provide other HUFAswhich are desired for fish larvae and juveniles, such as EPA and otherfish-characteristic n-3 HUFAs such as 18:3, 18:4, 20:4, and 22:5.

The inventors have found that such prey organisms for the method of theinvention can be obtained by enrichment with DHA enriched lipidmaterials, preferably materials derived from fish-based sources.

The prey organisms should preferably not contain too high content offree fatty acids, as these are generally considered harmful in largeamounts to fish larvae and juveniles. Preferably, free fatty acids areless than about 10 wt % of total lipid of the prey organisms accordingto the method of the invention.

In the present context, the expression ‘prey organisms’ refers to anymarine organism which can be used as live feed for larvae of aquaticspecies which are produced in aquacultural facilities. A general reviewof such prey organisms can be found in Lavens & Sorgeloos (eds.) “Manualon the production and use of live food for aquaculture” published by FAO(1995) which is hereby incorporated by reference. Accordingly, the mostcommonly used prey organisms include several classes and genera ofmicroalgae, rotifers, Artemia, zooplankton including copepods,cladocerans, nematodes, and trochophora larvae.

As used herein, the term ‘aquatic species’ is to be understood in itsbroadest sense and comprises both limnobiotic and marine species,including fish species such as salmon, trout carp, bass, bream, turbot,sea bass, sole, milkfish, gray mullet, grouper, sea bream, halibut,flounder, Japanese flounder, and monkfish; crustaceans such as shrimp,lobster, crayfish and crabs; and molluses such as bivalves.

A common feature of these aquatic species is that the life cycleincludes one or more larval stages which may have very specificnutritional requirements and accordingly the provision of live preyorganisms meeting this requirement is an essential factor for successfulaquacultural production. As mentioned above, one such specificrequirement is a high content of the essential fatty acid DHA, the term‘essential’ implying that the prey organisms are not capable of de novosynthesis of such compounds.

A particular embodiment of the invention involves the rearing of halibutspecies, however, the invention also encompasses other aquatic speciesas, for example, those mentioned above.

A primary benefit of the invention is that much higher survival ratesfor fish larvae can be achieved than have hitherto been reached by anyother methods, for species like Halibut and many others, to the best ofour knowledge. As demonstrated in the accompanying examples, consistentsurvival rates for Halibut during the larval stage of 65-80% and evenhigher, can be achieved in large scale fish farming according to theinvention, and other quality parameters such as correct pigmentation areexcellent

In a useful embodiment of the invention, the prey organisms are fed tothe aquatic species such as halibut, at a larval stage, preferably suchthat at least 30% of the larvae are alive at the end of the larvalstage, more preferably such that at least 50% are alive at the end ofthe larval stage including at least 60%, even more preferably such thatat least 70% are alive at the end of the larval stage, including that atleast 80% are alive at the end of the larval stage.

Correct pigmentation of the grown cultivated species is an importantfeature with respect to market value. A normally pigmented halibut has acolored ocular side and a white, non-pigmented blind side. In apreferred embodiment of the invention, at least 70% of halibut juveniles(newly metamorphosed larvae) show correct pigmentation, more preferablyat least 80%, even more preferably at least 90%, such as 95%, includingthat essentially all juveniles show correct pigmentation.

In certain embodiments, the invention provides a method of rearingaquatic species raised not primarily for consumption, such as ornamentalfish species and aquarium fish species.

According to the method, the prey organisms for feeding the aquaticspecies may be selected from any prey organisms that can be raised andused in aquaculture, in useful embodiments the prey organisms are acrustacean species such as Artemia, Copepoda, Daphnia, or Moina species;a Rotifera species including Brachionus plicatilis, Brachionusrotundiformis, and Brachionus rubens; or a Brachiopoda species.

The embodiments involving Artemia species are particularly useful. TheArtemia species can be cultivated and used according to the method ofthe invention at a naupliar, metanaupliar, or adult stage.

In certain embodiments of the invention, the aquatic organisms arereared in a marine environment. A marine environment is used herein todescribe an aqueous medium comprising sea water or simulating sea water,such as an aqueous medium with added salt, e.g., sodium chloride.

According to the invention, the prey organisms may be fed to the aquaticorganisms in any suitable form, e.g., as a composition comprising theprey organisms. In a preferred embodiment, the organisms of saidcomposition have a content of DHA of at least 20 wt % of the total lipidcontent of the organisms, more preferably at least 25 wt % of the totallipid content of the organisms, such as at (east 30 wt % of the totallipid content of the organisms.

In an embodiment of the invention, the composition comprises aquaticfish feed organisms having a content of DHA of at least 12 wt % of thetotal lipid content of the organisms, said composition comprising anaqueous phase of at least 50 wt %. In a particular embodiment, theaqueous phase of the composition comprises at least 0,5 wt % of sodiumchloride.

The composition is in other embodiments partially dry, such that is hasa water content less-than 50 wt %, such as at the most 40 wt %,including at the most 25 wt %, such as at the most 10 wt %, including atthe most 5 wt % of water. Such a composition may be in any suitableform, including a powder form, granules, and in the form of flakes.

As can be inferred from the above, in order for the composition toprovide a significant DHA ration, the total lipid content of the preyorganisms needs to be significant such as, e.g., at least 20 wt % on adry matter basis, including at least 25% dw, and preferably at least 30%dw.

EXAMPLE 1 Preparation of an Enrichment Composition for Fish Larvae PreyOrganisms

A composition for prey organisms such as Artemia species was prepared bycombining and mixing the following ingredients:

TABLE 1.1 phospholipid-rich component from squid mantles 9.7 g TG 4010(TM), Croda, essentially triglycerides w/ ≈40 wt % 78.0 g DHA vitamin C(ascorbyl palmitate) 8.5 g co-emulsifer, BASF Chremophore A25 (TM) 1.6 gGlucan Macroguard (TM) (immunostimulant) 0.8 g vitamin A (vitamin Apalmitate, 1 mill i.u./g) 0.190 g vitamin E (DL-alpha tocopherolacetate) 0.155 g vitamin B (thiamine hydrochloride) 1.2 g TBHQ(antioxidant) 0.036 g Ethoxyquin (antioxidant) 0.038 g Total 100 g

The phospholipid-rich component is prepared accordingly: Minced squid(150 kg) was added to 300 L of isopropanol and the mixture was agitatedrather vigorously for 4-6 h and left to stand overnight. Subsequently,the mixture was filtered and 300 L of hexane were added to the filtrateand mixed. This resulted in two phases which were allowed to separate.The upper phase, which largely consisted of hexane and isopropanol wasseparated and subjected to distillation in several rounds in vacuumusing a 50 L rotary evaporator to yield a total of 2.2 kg of aphospholipid enriched fraction as a brown-yellowish wax having aphospholipid content of about 65 wt % and a total DHA content of about40 wt %.

The TG 4010 material used as a DHA-rich component in the composition isderived from fish oil-based material which is enriched for DHA, itcomprises 40 wt % DHA, about 10 wt % EPA and about 10 wt % other n-3HUFAs. The fatty acids are mostly in the form of triglycerides and thematerial has a very low free fatty acid content.

Other materials have been tested as sources of a DHA-rich component,such as TG 5010 (also from Croda) which has a DHA content of about 50 wt%, and enzymatically synthesized highly DHA-enriched triglycerides.

EXAMPLE 2 Cultivation of Enriched Artemia for Rearing of Aquatic Species

Artemia cysts were hatched under optimal conditions (in seawater, 27-29°C., pH about 8, oxygen content above 4 mg/L). The newly hatched naupliarArtemia were rinsed and put in 250 L tanks to give a density of200.000/L. Temperature was kept at 25-28° C., oxygen content at 5-6 mg/Land pH buffered at 7.5 with sodium bicarbonate (2 g/L). The tanks wereaerated by passing atmospheric air through perforated hoses at bottom oftanks. Enrichment composition as described in Example 1 was added to thetanks to a concentration of 0,2 g/L and the same amount added 10 hlater. 24 h after the first addition of enrichment composition theArtemia has the following lipid composition (31 % dw (dry weight)lipids, numbers in left-most column refer to the number of carbons anddouble bond In the fatty acids of the lipid components, DHA is 22:6 andEPA 20:5):

PL TG FFA Total 16% 76% 8% 100% 14:0 8.8 1.0 3.1 0.8 16:0 15.0 8.8 36.011.1 16:1 2.6 3.2 3.1 2.5 18:0 6.4 2.7 6.3 4.2 18:1 25.2 15.6 13.0 17.118:2 4.2 3.5 1.8 3.3 18:3 13.2 19.2 6.5 14.7 18:4 2.2 3.1 1.7 2.4 20:11.6 1.0 0.0 0.9 20:4 2.8 2.1 0.0 2.2 20:5 12.5 10.2 4.4 9.5 22:1 0.0 0.00.0 22:4 0.0 1.1 0.0 1.2 22:5 0.0 1.0 0.0 1.1 22:6 4.6 20.0 14.8 18.999.0 92.5 90.7 90.0

The Artemia thus obtained has a highly enriched total concentration ofDHA and is very suitable for feeding fish larvae such as halibut larvaeaccording to the invention.

EXAMPLE 3 Cultivation of Artemia with Alternative Enrichment Composition

Newly hatched Artemia wer placed in 250 L tanks and same conditions asdescribed in Example 2. The Artemia were fed a lipid composition mixed 2wt % Chremophore A25 emulsifier. The lipid composition contained 50 wt %of the phospholipid component of the Example 1 composition; 25 wt %‘DHA-80’, essentially triglycerides comprising 80 wt % DHA, synthesizedenzymatically from glycerol and DHA fatty acid using lipase from CandidaAntarctica (as described in U.S. Pat. No. 5,604,119); and 25 wt %Lysi-22 (TM) (Lysi hf, Iceland), a fish oil with 22 wt % DHA. The feedcomposition was added to the tanks to a concentration of 0,2 g/L and thesame amount added 12 h later. 24 h after the first addition ofenrichment composition the Artemia has the following lipid composition(34% dw lipids):

PL TG FFA Total 25% 72% 3% 100% 14:0 0.9 1.1 0.0 1.3 16:0 13.6 10.6 32.011.2 16:1 3.3 3.5 3.3 3.4 18:0 5.8 2.2 10.6 3.3 18:1 26.2 15.4 15.7 15.118:2 3.7 2.7 0.0 2.5 18:3 13.8 15.0 4.4 13.7 18:4 2.7 2.1 0.0 2.2 20:11.0 1.9 5.3 2.0 20:4 2.1 1.7 0.0 1.9 20:5 13.1 8.7 5.0 9.7 22:6 8.4 28.823.6 28.0 94.6 93.7 100.0 94.1

The Artemia obtained has a very highly enriched total concentration ofDHA (9,5% dw) in accordance with the invention as well as otherfish-characteristic n-3 HUFAs, and is thus particularly suitable forfeeding fish larvae such as halibut larvae.

EXAMPLE 4 Use of Enrichment Composition for Cultivating Rotifers(Brachionus Plicatilis)

Rotifers were reared under similar conditions as described in Example 2,they were fed with Isochrysis plankton and yeast and enriched for 6 h at27 C with an enrichment composition as described in Example 2, exceptthat Croda 50 was used instead of Croda 40, Croda 50 containing about 50wt % of DHA. The rotifers had the following lipid composition (22% dwlipids):

PL TG FFA Total 32% 56% 13% 100% 14:0 6.6 7.8 3.3 6.9 16:0 25.9 4.9 15.213.0 16:1 1.9 2.5 1.3 2.2 18:0 3.6 5.7 2.7 4.7 18:1 4.5 4.5 5.5 4.7 18:24.9 0.3 2.0 2.0 18:3 3.1 3.2 1.9 3.0 18:4 2.2 6.2 2.2 4.4 20:1 1.2 1.91.5 1.6 20:4 5.0 2.3 2.2 3.2 20:5 10.1 14.7 14.8 13.4 22:6 25.6 38.840.4 35.2 94.7 92.7 93.0 94.3

The rotifers obtained have a very high total concentration of DHA, and avery high phospholipid content and thus and thus are highly suitable foraquacultural rearing according to the invention.

EXAMPLE 5 Use of HUFA- and Phospholipid-enriched Artemia forAquacultural Rearig of Halibut

Halibut larvae were first fed at 230-250° d. (‘° d’ multiplicationfactor of temperature (° C.) and days since hatching.) Circular rearingtanks were used, either 3,5 or 7 m³. Larvae were gradually acclimatizedto a rearing temperature of 11° C. and a light intensity of 300-500 lux.The larvae were fed Artemia twice per day, in the morning and in thelate afternoon. The Artemia was enriched with an enrichment composition24 h before the morning feed, then stored at 13-15° C. for another 7-8 hfor the afternoon feed. Feed rations were adjusted to allow for gooddigestion of the Artemia. Microalgae (Isocrysis sp.) wee added to therearing water to reduce stress and facilitate maximum ingestion rates.Slight aeration was applied in the center of the tanks to homogenize thewater quality and the feed particles. Slight circular current wasacquired with the inflow to distribute the larvae. Water exchange wasincreased from 1,2 times per 24 h in the beginning up to 3,3 times per24 h in the end. Larval rearing tanks were cleaned daily.

Survival rates of over 80% in one tank from start of feed to end oflarval stage were observed (90% excluding “gapers”: larvae with jawdeformity), and frequently survival rates between 65 and 75% have beenobserved. On average about 80% of juveniles showed correct pigmentation,but upto 96% correct pigmentation in one tank were observed. Correctpigmentation is defined as a normal pigmentation color on the ocularside and no pigmentation on the blind side. About 65% of juveniles onaverage but up to 80% in one tank showed correct eye migration, that ishaving both eyes on the ocular side. Ongoing experiments indicate thateven higher average survival and pigmentation rates are obtainable.

The results show that DHA-enriched prey organisms according to theinvention are particularly suitable for the rearing of aquatic speciessuch as halibut in terms of high survival rates and quality.

1. A method of rearing an aquatic species, the method comprising feeding the aquatic species during at least part of the larval and/or post-larval stage with prey organisms, the prey organisms having in their total lipid content a content of DHA of at least 12 wt %; providing n-3 HUFAs including 18:3, 18:4, 20:4, 20:5 (EPA) and 22:5 fatty acids, the organisms comprising in the range of 5-35 wt % phospholipids of the total lipid content, and having a total lipid content of at least 20 wt % on a dry weight basis.
 2. A method according to claim 1 wherein the DHA content in the total lipid content is at least 20 wt %.
 3. A method according to claim 1 wherein the prey organisms are cultivated and are selected from the group consisting of a crustacean species, Rotifera species and Brachiopoda species.
 4. A method according to claim 3 wherein the crustacean species is an Artemia species.
 5. A method according to claim 4 wherein the Artemia species is cultivated in the aqueous medium at a naupliar, metanaupliar, or adult stage.
 6. A method according to claim 3 wherein the prey organisms are Rotifera species or a Brachiopoda species.
 7. A method according to claim 6 wherein the Rotifera species includes Brachionus plicatilis, Brachionus rotundiformis, and/or Brachionus rubens.
 8. A method according to claim 1 wherein the aquatic species being reared is of a halibut species.
 9. A method according to claim 8 wherein the prey organisms are fed to the halibut species at its larval stage.
 10. A method according to claim 9 wherein at least 30% of the larvae being fed are alive at the end of the larval stage.
 11. A method according to claim 10 wherein at least 50% of the larvae being fed are alive at the end of the larval stage.
 12. A method according to claim 11 wherein at least 70% of the larvae being fed are alive at the end of the larval stage.
 13. A method according to claim 9 wherein after metamorphosis at least 70% of juveniles show correct pigmentation.
 14. A method according to claim 1 wherein at least 90% of juveniles show correct pigmentation.
 15. A method according to claim 1 wherein the aquatic species is reared in a marine environment.
 16. A method according to claim 1 wherein the aquatic species is fed an the feed composition comprising aquatic prey organisms having a content of DHA of at least 20 wt % of the total lipid content of the prey organisms.
 17. A method according to claim 16 wherein the aquatic species is fed a composition comprising the prey organisms having a content of DHA of at least 25 wt % of the total lipid content of the prey organisms.
 18. A method according to claim 1 wherein the aquatic species is fed a composition comprising aquatic fish feed organisms comprising the prey organisms, the composition comprising an aqueous phase of at least 50 wt %.
 19. A method according to claim 18 wherein the aqueous phase comprises at least 0.5 wt % of NaCl.
 20. A method according to claim 1 wherein the aquatic species is fed a composition comprising aquatic feed animals comprising the prey organisms, the composition having a water content that is less than 50 wt %.
 21. A method according to claim 20 wherein the composition is in a form selected from the group consisting of powder, granules and flakes.
 22. A method according to claim 1 wherein the aquatic species being fed is selected from the group consisting of fish, crustaceans and molluses.
 23. A method according to claim 1 wherein the aquatic species being fed is selected from the group consisting of an ornamental fish species and an aquarium fish species.
 24. A method according to claim 1, wherein the DHA content in the total lipid content is at least 25 wt %.
 25. A method according to claim 3, wherein the crustacean species include Artemia, Copepoda, Daphnia, and Moina species.
 26. A method according to claim 9, wherein after metamorphoses at least 80% of juveniles show correct pigmentation.
 27. A method according to claim 1, wherein after metamorphoses at least 95% of juveniles show correct pigmentation.
 28. A method according to claim 1, wherein the prey organisms are In C. composition having a water content of not more than 10 wt %.
 29. A method according to claim 22, wherein the fish includes salmon, trout, carp, bass, bream, turbot, sole, milkfish, grey muller, grouper, flounder, sea bass, cod, haddock, Japanese flounder and/or eel; the crustaceans include shrimp, lobster, crayfish and crabs; and the molluses include bivalves. 